Brain fractalkine-CX3CR1 signalling is anti-obesity system as anorexigenic and anti-inflammatory actions in diet-induced obese mice

Fractalkine is one of the CX3C chemokine family, and it is widely expressed in the brain including the hypothalamus. In the brain, fractalkine is expressed in neurons and binds to a CX3C chemokine receptor 1 (CX3CR1) in microglia. The hypothalamus regulates energy homeostasis of which dysregulation is associated with obesity. Therefore, we examined whether fractalkine-CX3CR1 signalling involved in regulating food intake and hypothalamic inflammation associated with obesity pathogenesis. In the present study, fractalkine significantly reduced food intake induced by several experimental stimuli and significantly increased brain-derived neurotrophic factor (BDNF) mRNA expression in the hypothalamus. Moreover, tyrosine receptor kinase B (TrkB) antagonist impaired fractalkine-induced anorexigenic actions. In addition, compared with wild-type mice, CX3CR1-deficient mice showed a significant increase in food intake and a significant decrease in BDNF mRNA expression in the hypothalamus. Mice fed a high-fat diet (HFD) for 16 weeks showed hypothalamic inflammation and reduced fractalkine mRNA expression in the hypothalamus. Intracerebroventricular administration of fractalkine significantly suppressed HFD-induced hypothalamic inflammation in mice. HFD intake for 4 weeks caused hypothalamic inflammation in CX3CR1-deficient mice, but not in wild-type mice. These findings suggest that fractalkine-CX3CR1 signalling induces anorexigenic actions via activation of the BDNF-TrkB pathway and suppresses HFD-induced hypothalamic inflammation in mice.


Effects of intracerebroventricular administration of fractalkine on food intake in mice.
Food intake during 4-h refeeding after 48-h fasting was significantly suppressed by intracerebroventricular administration of fractalkine in a dose-dependent manner (3 and 10 μg/mouse) compared with saline treatment (Fig. 2a). Intracerebroventricular administration of fractalkine (3 μg/mouse) 1 h before the start of the dark phase significantly suppressed nocturnal food intake compared with saline treatment (Fig. 2b). Compared with saline treatment, concomitant intracerebroventricular administration of fractalkine (3 μg/mouse) with NPY (20 μg/mouse) significantly suppressed the food intake induced by NPY (Fig. 2c). In addition, intracerebroventricular administration of fractalkine (3 μg/mouse) significantly suppressed food intake induced by intraperitoneal administration of ghrelin (360 μg/kg) (Fig. 2d).

Involvement of BDNF in the anorexigenic effect of fractalkine in mice.
Intracerebroventricular administration of fractalkine (3 μg/mouse) significantly increased BDNF mRNA expression in the hypothalamus of normal mice to 143% of saline treatment (Fig. 3a). In addition, intracerebroventricular administration of fractalkine (3 μg/mouse) significantly increased BDNF mRNA expression in the hypothalamus of mice after 4-h refeeding to 146% of saline treatment (Fig. 3b). On the other hand, intracerebroventricular administration of fractalkine (3 μg/mouse) did not change the mRNA expression of NPY, AgRP, orexin, melanin-concentrating hormone (MCH), POMC, or CART in the hypothalamus of normal or refed mice (Fig. 3a, b). Moreover, application of fractalkine (100 nM) for 3 h significantly increased BDNF mRNA expression in MG6 cells, a mouse microglia cell line (Fig. 3c). To examine the involvement of BDNF in the anorectic effect of fractalkine, the TrkB antagonist ANA-12 (0.5 mg/kg) was intraperitoneally administrated at 30 min before intracerebroventricular administration of fractalkine (3 μg/mouse). ANA-12 significantly attenuated the suppressive actions of fractalkine, as well as BDNF, on food intake during 4-h refeeding after 48-h fasting (Fig. 3d).
Effects of intracerebroventricular administration of fractalkine on increased mRNA expression of TNF-α and IL-6 in the hypothalamus of mice fed a HFD. Fractalkine has anti-inflammatory actions [20][21][22][23][24] . Therefore, we examined the effects of intracerebroventricular administration of fractalkine on increased proinflammatory cytokine expression in the hypothalamus of mice fed a HFD for 16 weeks. The intracerebroventricular administration of fractalkine (3 μg/mouse) significantly decreased the increased mRNA expression of TNF-α and IL-6 in the hypothalamus of mice fed a HFD for 16 weeks by 65 and 64% as compared with saline-treated mice fed a HFD, respectively (Fig. 6a, b).  www.nature.com/scientificreports/ Changes in the mRNA expression of TNF-α and IL-6 in the hypothalamus and cumulative food intake of CX3CR1-deficient mice fed a HFD. In CX3CR1-deficient mice, HFD feeding for 4 weeks, but not 2 weeks, significantly increased TNF-α and IL-6 mRNA expression in the hypothalamus to 138% and 143%, respectively, compared with CD feeding, while wild-type mice did not (Fig. 7a, b). In the CD-feeding groups, TNF-α and IL-6 mRNA expression in the hypothalamus was not different between CX3CR1-deficient mice and wild-type mice (Fig. 7a, b). In both wild-type and CX3CR1-deficient mice, HFD feeding for more than a week induced a significant increase in body weight compared with CD feeding (Fig. 7c). Body weight in CX3CR1deficient mice fed a HFD was significantly increased by HFD feeding for more than 3 weeks compared with that in wild-type mice fed a HFD (Fig. 7c). Mesenteric fat and epididymal fat were significantly increased by HFD feeding for more than 2 weeks in both wild-type and CX3CR1-deficient mice compared with that in CD feeding (Fig. 7d, e). CX3CR1-deficient mice fed a HFD for 4 weeks showed a significant increase in mesenteric fat, but not epididymal fat, compared with wild-type mice fed a HFD (Fig. 7d, e). Cumulative food intake of both the CD and HFD for 2 and 4 weeks in CX3CR1-deficient mice was significantly increased compared with that in wild-type mice (Fig. 7f).

Discussion
The present study demonstrated that fractalkine suppresses food intake via activation of the BDNF-TrkB pathway and that reduction of fractalkine-CX3CR1 signalling exacerbates HFD-induced hypothalamic inflammation and accelerates obesity in HFD-fed mice (Fig. 7g). The hypothalamus is considered an important region for the regulation of food intake. Consistent with previous report 25 , the present study showed that 48 h of fasting significantly increased orexigenic peptides and decreased anorexigenic factors in the hypothalamus. Fasting decreases plasma leptin, insulin, and glucose, which are involved in the regulation of gene expression in the hypothalamus. However, it is difficult to determine the independent role of each factor in the regulation of hypothalamic gene expression because leptin, insulin, and glucose tend to form regulatory loops with each other and co-vary. In the present study, we showed that mRNA expression of MC3R, MC4R, and TrkB, the receptor for BDNF, which acts as a downstream effector of MC4R  www.nature.com/scientificreports/ neuroendocrine, and autonomic pathways in response to acute changes in energy stores. MC4R is well known to act as a master regulator of body weight set point 26,27 . In contrast to the MC4R, deletion of the MC3R does not produce measurable hyperphagia or hypometabolism under normal conditions. However, MC3R regulates normal fasting response and adaptation to restricted feeding 28 . In the present study, fractalkine and CX3CR1 mRNA expression in the hypothalamus was altered after fasting-feeding. Therefore, we speculated that fractalkine-CX3CR1 signalling may act to modulate feeding.
In the present study, intracerebroventricular administration of fractalkine significantly reduced food intake during 4-h refeeding after 48-h fasting, nocturnal food intake, and food intake induced by NPY and ghrelin. A previous study showed that BDNF in the PVN inhibits deprivation-induced feeding, normal dark-phase feeding, and NPY-induced feeding 29 . Therefore, we considered the involvement of BDNF in the anorexigenic mechanisms of fractalkine. In the present study showed that intracerebroventricular administration of fractalkine significantly increased BDNF mRNA expression in the hypothalamus of mice. Moreover, the TrkB antagonist, ANA-12 significantly attenuated the suppressive actions of fractalkine on food intake during 4-h refeeding after 48-h fasting. The anorexigenic actions of BDNF are mediated by the high-affinity receptor TrkB, and mutations in BDNF or TrkB genes may account for certain types of obesity or other forms of eating disorders in humans 30 . Since there is little co-expression of TrkB and CART or NPY in the hypothalamic nucleus, it is unlikely that BDNF directly regulates POMC/CART or NPY/AgRP-expressing neurons in the ARC. However, there is some evidence for a link between melanocortin signalling and BDNF-TrkB signalling, suggesting that BDNF acts as a downstream effector of MC4R signalling to regulate energy balance 31 . BDNF is thought to interact with NPY to exert its anorexigenic effects 29 , but its direct action on NPY has not yet been established. Based on these findings and the present study, it is likely that BDNF acts downstream of the actions of these peptides and that fractalkine suppresses food intake via activation of BDNF-TrkB signalling without altering mRNA expression of other feeding regulatory peptides. Because CX3CR1 is only expressed on microglia, we used the microglia cell line MG6 cells to examine the effects of fractalkine on BDNF mRNA expression in an in vitro experiment. Application of fractalkine significantly increased BDNF mRNA expression in MG6 cells. However, BDNF is expressed in not only microglia but also neurons 32 . Stimulation of microglial cells by fractalkine induces an increase in extracellular adenosine levels 33 . Activation of the adenosine receptor A 2A receptor with the A 2A receptor agonist CGS21680 increases BDNF expression in developing rat primary cortical neurons 32 and murine N9 microglial cells 34 . These reports [32][33][34] suggest that the increase in hypothalamic BDNF mRNA expression induced by intracerebroventricular administration of fractalkine may be induced in neurons as well as microglia. Furthermore, we examined food intake and hypothalamic BDNF mRNA expression in CX3CR1-deficient mice. CX3CR1-deficient mice showed an increase in cumulative food intake for 3 days and a decrease in hypothalamic BDNF mRNA expression compared with wild-type mice. These findings suggest that fractalkine-CX3CR1 signalling has anorexigenic actions that are mediated by an increase in BDNF which is an anorexigenic peptide in the hypothalamus.
Obesity results from the dysregulation of energy metabolism 35 . In both human and animal models, dietinduced obesity is associated with increased circulating inflammatory markers such as TNF-α, IL-1β, and IL-6, which represent a key step in the development of insulin resistance in peripheral organs 36,37 . Fractalkine is widely present not only in the brain but also in peripheral tissues such as the heart, lung, kidney, muscle, testis, and adipose tissue 38 . CX3CR1-deficient mice exhibit glucose intolerance mainly due to beta cell dysfunction, while fractalkine treatment improves glucose tolerance and increases insulin secretion in wild-type mice 39 . Moreover, deficient fractalkine-CX3CR1 signalling exacerbates diet-induced insulin resistance, hepatic steatosis, and adipose tissue inflammation 40 . Furthermore, just as in peripheral tissues, in animal models of genetic and diet-induced obesity, inducing an inflammatory response in the hypothalamus leads to the molecular and functional resistance to the adipostatic hormones leptin and insulin, resulting in defective control of food intake and energy expenditure 17,41,42 . Experimental interventions that block hypothalamic inflammation reduce food intake, leptin resistance, and body weight in animals exposed to HFD feeding [16][17][18]43,44 . Depleting microglia from the hypothalamus of mice abolishes inflammation and neuronal stress induced by excess saturated fatty acid www.nature.com/scientificreports/ consumption 44 . In this context, these findings indicate that microglia are potently involved in brain inflammation induced by HFD feeding 44 . De Souza demonstrated that immune-related molecules, including proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, in the hypothalamus significantly increase after 16 weeks of HFD feeding 45 . Consistent with this finding, the present study showed that HFD feeding for 16 weeks, but not 2, 4, and 8 weeks, resulted in a significant increase in mRNA expression of TNF-α and IL-6 in the hypothalamus of mice. Fractalkine levels are downregulated in the adipose tissue, plasma and brain in diet-induced obese mice 19,40,46 . The present study showed a significant decrease in the mRNA expression of fractalkine, but not CX3CR1, in the hypothalamus of mice fed a HFD for 16 weeks. Our previous report revealed that long-term HFD feeding in mice reduces fractalkine expression in the hippocampus and amygdala, which may be caused by decreases in plasma IGF-1 levels and brain BDNF levels induced by long-term HFD feeding 46 . Contrary to our results, previous study showed that HFD intake for 8 weeks increases fractalkine and CX3CR1 expression in the hypothalamus in male Swiss mice, whereas HFD intake does not change fractalkine and CX3CR1 expression in the hypothalamus of male Balb-c mice 47 . Moreover, another paper reported that male C57BL/6 J mice fed HFD for 18 weeks shows www.nature.com/scientificreports/ a decrease in fractalkine and CX3CR1, but not female 19 . These differences may relate to strain (Swiss mice are extremely DIO-sensitive) and sex. Previous reports showed that exogenous fractalkine decreases TNF-α secretion induced by microglial activation 20 , while neutralization of endogenous brain fractalkine increases TNF-α production induced by lipopolysaccharide 22 . However, another report showed that inhibition of hypothalamic fractalkine reduces HFDinduced hypothalamic inflammation in male Swiss mice 47 . CX3CR1-deficient mice show excessive cellular activation and overproduction of inflammatory mediators, generally increasing susceptibility to CNS inflammatory diseases 48,49 . However, some studies have identified protective aspects of CX3CR1 deficiency 50 . These reports suggest that whether fractalkine promotes or protects inflammation depends on the state of inflammation and the conditions of the experiments, suggesting that the action of fractalkine-CX3CR1 signaling on inflammation is complexly regulated. In the present study, the intracerebroventricular administration of fractalkine significantly suppressed the increase in the mRNA expression of both TNF-α and IL-6 in the hypothalamus of mice fed a HFD for 16 weeks. Conversely, CX3CR1-deficient mice exhibited hypothalamic inflammation induced by HFD feeding for 4 weeks, whereas wild-type mice did not. Taken together, we hypothesized that reduced fractalkine-CX3CR1 signalling in the hypothalamus of mice fed a HFD may lead to exaggerated hypothalamic microglial activation and inflammatory responses induced by HFD consumption. In addition, body weight in CX3CR1-deficient mice fed a HFD was significantly increased by HFD feeding for more than 3 weeks compared with that in wild-type mice fed a HFD. However, contrary to our study, previous reports 40,51 demonstrated that CX3CR1-deficient mice fed a HFD show similar changes in body weight to wild-type mice fed a HFD. A possible explanation for this discrepancy may reside in the several differences in the methodological procedures as follows; (1) in the present study, HFD feeding started at 14-16 weeks old, while HFD feeding started at 4 weeks old in previous study 40 , (2) in the present study, HFD containing 60% fat of total calories was used, while HFD containing 45% fat of total calories was used in previous study 51 . It seems likely that these methodological differences may induce the different results in each report. In the present study, CX3CR1-deficient mice fed a HFD for 4 weeks showed a significant increase in mesenteric fat as well as body weight compared with wild-type mice fed HFD, that is, CX3CR1-deficient mice may have increased body weight and mesenteric fat due to excess energy intake from increased HFD feeding. The CX3CR1-deficient mice used in the present study were systemically deficient but may reflect, at least in part, the effects of defective central fractalkine-CX3CR1 signalling.
The present study demonstrated that fractalkine-CX3CR1 signalling has anorexigenic and anti-inflammatory actions in the hypothalamus of diet-induced obese mice. Of considerable interest for the present study is the possibility that CX3CR1 activation may be a promising way to reduce body weight and improve brain inflammation associated with obesity. Our study provides that fractalkine-CX3CR1 signalling may be an attractive target for the pharmacological treatment of obesity.

Methods
Animals. Male C57BL/6 J mice (6 weeks old) were obtained from CLEA Japan, Inc. (Tokyo, Japan). CX3CR1deficient mice, in which both copies of CX3CR1 were disrupted by the gene for enhanced green fluorescent protein 52 , were initially obtained from The Jackson Laboratory and then bred to maintain them on the C57BL/6 J background.
Mice were housed in plastic cages in a room kept at room temperature of 23 ± 1 ℃ with a 12: 12 h light-dark cycle (lights turned on at 7:00 A.M. ). Ad libitum access to water and food (CE-2; CLEA Japan, Inc.) was provided to all mice. The mice were used in the experiments at 6-16 weeks of age. All animal experiments complied with the ARRIVE guidelines and were performed in accordance with the guidelines established by the Institute of Laboratory Animal Science Research Support Center at Kagoshima University and approved by the Kagoshima University Institutional Animal Care and Use Committee (protocol nos. MD15060, MD16052、MD17060, and MD18079), and in accordance with the guidelines established by the United States National Institutes of Health

Intracerebroventricular injection. Intracerebroventricular injection was performed according to our
previous report 53  Nocturnal food intake. Male mice at 10-12 weeks of age were used in the experiments. To assess the effect of intracerebroventricular administration of fractalkine on nocturnal food intake, fractalkine (3 μg/2 μl/mouse) was injected intracerebroventricularly 1 h before the beginning of the dark phase. Mice in the control group were given intracerebroventricular administration of an equal volume of saline. Food intake was measured for 15 h after intracerebroventricular injection. Food and water were available ad libitum at all times.

Food intake induced by NPY and ghrelin.
Male mice at 10-12 weeks of age were used in the experiments. The experiments were performed from 11:00 A.M. to 3:00 P.M. Fractalkine (3 μg/2 μl/mouse) was intracerebroventricularly administered just before intracerebroventricular administration of NPY (20 μg/2 μl/mouse; dissolved in saline) or intraperitoneal administration of ghrelin (360 μg/kg, 10 ml/kg body weight; dissolved in saline). Mice in the control group were given an intracerebroventricular or intraperitoneal administration of an equal volume of saline. Food intake was measured for 4 h after peptide injection. Food and water were available ad libitum at all times.
Intraperitoneal administration of ANA-12. ANA-12 was dissolved in dimethyl sulfoxide (DMSO), and diluted with saline in a final concentration of 2% DMSO. Male mice at 10-12 weeks of age were used in the experiments. ANA-12 (0.5 mg/kg, 10 ml/kg body weight) was intraperitoneally administered after 48-h fasting.
In the vehicle treatment groups, an equal volume of vehicle (2% DMSO saline) was intraperitoneally administered. Fractalkine (3 μg/2 μl/mouse) and BDNF (0.5 μg/2 μl/mouse) were intracerebroventricularly administered 30 min after the administration of ANA-12 and then food intake was measured for 4 h after peptide injection. Food and water were available ad libitum during the experiments.
Food intake of CX3CR1-deficient mice. Male CX3CR1-deficient mice at 10-12 weeks of age were used in the experiments. Cumulative food intake was measured for 3 days. Food and water were available ad libitum during the experiments. At the end of experiments, the hypothalamus was collected to examine BDNF mRNA expression under isoflurane anesthesia 54 .
Analysis in mice fed a HFD. Normal male mice (6 weeks old) were randomly divided into two groups. The first group was given CE-2 as a CD containing 12.6% fat of total calories (343.1 kcal/100 g). The second group was given a HFD (no. D12492; Research Diets, Inc., New Brunswick, NJ) containing 60% fat of total calories, predominantly in the form of lard (524 kcal/100 g). After feeding on the diets for 2, 4, 8, or 16 weeks, the hypothalamus was collected under isoflurane anesthesia. Fractalkine was dissolved in saline. The mice fed a HFD for 16 weeks were killed at 3 h after the intracerebroventricular administration of fractalkine (3 μg/mouse), and the hypothalamus was collected under isoflurane anesthesia. In the control group, an equal volume of saline (2 μl/mouse) was intracerebroventricularly administered.
Male CX3CR1-deficient mice (14-16 weeks old) were randomly divided into two groups. The first group was given a CD. The second group was given a HFD. After feeding on the diets for 2 or 4 weeks, the hypothalamus, mesenteric fat, and epididymal fat were collected under isoflurane anesthesia.
Sampling of the hypothalamus. Mice were killed by decapitation. The hypothalamus was rapidly removed from the skull and placed on an ice-cooled paraffin plate for dissection of the hypothalamus as previously described 54 . The hypothalamus was immediately frozen in liquid nitrogen and stored at − 80 °C until analyzed.